Emc1 is essential for vision and zebrafish photoreceptor outer segment morphogenesis.

Inherited retinal diseases (IRDs) are a rare group of eye disorders characterized by progressive dysfunction and degeneration of retinal cells. In this study, we characterized the raifteirí (raf) zebrafish, a novel model of inherited blindness, identified through an unbiased ENU mutagenesis screen. A mutation in the largest subunit of the endoplasmic reticulum membrane protein complex, emc1 was subsequently identified as the causative raf mutation. We sought to elucidate the cellular and molecular phenotypes in the emc1-/- knockout model and explore the association of emc1 with retinal degeneration. Visual behavior and retinal electrophysiology assays demonstrated that emc1-/- mutants had severe visual impairments. Retinal histology and morphometric analysis revealed extensive abnormalities, including thinning of the photoreceptor layer, in addition to large gaps surrounding the lens. Notably, photoreceptor outer segments were drastically smaller, outer segment protein expression was altered and hyaloid vasculature development was disrupted. Transcriptomic profiling identified cone and rod-specific phototransduction genes significantly downregulated by loss of emc1. These data shed light on why emc1 is a causative gene in inherited retinal disease and how outer segment morphogenesis is regulated.

The vitamin B12 processing enzyme, mmachc, is essential for zebrafish survival, growth and retinal morphology.

Cobalamin C (cblC) deficiency, the most common inborn error of intracellular cobalamin metabolism, is caused by mutations in MMACHC, a gene responsible for the processing and intracellular trafficking of vitamin B12. This recessive disorder is characterized by a failure to metabolize cobalamin into adenosyl- and methylcobalamin, which results in the biochemical perturbations of methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia caused by the impaired activity of the downstream enzymes, methylmalonyl-CoA mutase and methionine synthase. Cobalamin C deficiency can be accompanied by a wide spectrum of clinical manifestations, including progressive blindness, and, in mice, manifests with very early embryonic lethality. Because zebrafish harbor a full complement of cobalamin metabolic enzymes, we used genome editing to study the loss of mmachc function and to develop the first viable animal model of cblC deficiency. mmachc mutants survived the embryonic period but perished in early juvenile life. The mutants displayed the metabolic and clinical features of cblC deficiency including methylmalonic acidemia, severe growth retardation and lethality. Morphologic and metabolic parameters improved when the mutants were raised in water supplemented with small molecules used to treat patients, including hydroxocobalamin, methylcobalamin, methionine and betaine. Furthermore, mmachc mutants bred to express rod and/or cone fluorescent reporters, manifested a retinopathy and thin optic nerves (ON). Expression analysis using whole eye mRNA revealed the dysregulation of genes involved in phototransduction and cholesterol metabolism. Zebrafish with mmachc deficiency recapitulate the several of the phenotypic and biochemical features of the human disorder, including ocular pathology, and show a response to established treatments.

Maf-family bZIP transcription factor NRL interacts with RNA-binding proteins and R-loops in retinal photoreceptors.

RNA-binding proteins (RBPs) perform diverse functions including the regulation of chromatin dynamics and the coupling of transcription with RNA processing. However, our understanding of their actions in mammalian neurons remains limited. Using affinity purification, yeast-two-hybrid and proximity ligation assays, we identified interactions of multiple RBPs with NRL, a Maf-family bZIP transcription factor critical for retinal rod photoreceptor development and function. In addition to splicing, many NRL-interacting RBPs are associated with R-loops, which form during transcription and increase during photoreceptor maturation. Focusing on DHX9 RNA helicase, we demonstrate that its expression is modulated by NRL and that the NRL-DHX9 interaction is positively influenced by R-loops. ssDRIP-Seq analysis reveals both stranded and unstranded R-loops at distinct genomic elements, characterized by active and inactive epigenetic signatures and enriched at neuronal genes. NRL binds to both types of R-loops, suggesting an epigenetically independent function. Our findings suggest additional functions of NRL during transcription and highlight complex interactions among transcription factors, RBPs, and R-loops in regulating photoreceptor gene expression in the mammalian retina.

Selective transcriptomic dysregulation of metabolic pathways in liver and retina by short- and long-term dietary hyperglycemia.

A high glycemic index (HGI) diet induces hyperglycemia, a risk factor for diseases affecting multiple organ systems. Here, we evaluated tissue-specific adaptations in the liver and retina after feeding HGI diet to mice for 1 or 12 month. In the liver, genes associated with inflammation and fatty acid metabolism were altered within 1 month of HGI diet, whereas 12-month HGI diet-fed group showed dysregulated expression of cytochrome P450 genes and overexpression of lipogenic factors including Srebf1 and Elovl5. In contrast, retinal transcriptome exhibited HGI-related notable alterations in energy metabolism genes only after 12 months. Liver fatty acid profiles in HGI group revealed higher levels of monounsaturated and lower levels of saturated and polyunsaturated fatty acids. Additionally, HGI diet increased blood low-density lipoprotein, and diet-aging interactions affected expression of mitochondrial oxidative phosphorylation genes in the liver and disease-associated genes in retina. Thus, our findings provide new insights into retinal and hepatic adaptive mechanisms to dietary hyperglycemia.

Incomplete splicing, cell division defects, and hematopoietic blockage in dhx8 mutant zebrafish.

BACKGROUND: Vertebrate hematopoiesis is a complex developmental process that is controlled by genes in diverse pathways. To identify novel genes involved in early hematopoiesis, we conducted an ENU (N-ethyl-N-nitrosourea) mutagenesis screen in zebrafish. The mummy (mmy) line was investigated because of its multiple hematopoietic defects. RESULTS: Homozygous mmy embryos lacked circulating blood cell types and were dead by 30 hr post-fertilization (hpf). The mmy mutants did not express myeloid markers and had significantly decreased expression of progenitor and erythroid markers in primitive hematopoiesis. Through positional cloning, we identified a truncation mutation in dhx8 in the mmy fish. dhx8 is the zebrafish ortholog of the yeast splicing factor prp22, which is a DEAH-box RNA helicase. mmy mutants had splicing defects in many genes, including several hematopoietic genes. mmy embryos also showed cell division defects as characterized by disorganized mitotic spindles and formation of multiple spindle poles in mitotic cells. These cell division defects were confirmed by DHX8 knockdown in HeLa cells. CONCLUSIONS: Together, our results confirm that dhx8 is involved in mRNA splicing and suggest that it is also important for cell division during mitosis. This is the first vertebrate model for dhx8, whose function is essential for primitive hematopoiesis in developing embryos.

Long wavelength-sensing cones of zebrafish retina exhibit multiple layers of transcriptional heterogeneity.

Introduction: Understanding how photoreceptor genes are regulated is important for investigating retinal development and disease. While much is known about gene regulation in cones, the mechanism by which tandemly-replicated opsins, such as human long wavelength-sensitive and middle wavelength-sensitive opsins, are differentially regulated remains elusive. In this study, we aimed to further our understanding of transcriptional heterogeneity in cones that express tandemly-replicated opsins and the regulation of such differential expression using zebrafish, which express the tandemly-replicated opsins lws1 and lws2. Methods: We performed bulk and single cell RNA-Seq of LWS1 and LWS2 cones, evaluated expression patterns of selected genes of interest using multiplex fluorescence in situ hybridization, and used exogenous thyroid hormone (TH) treatments to test selected genes for potential control by thyroid hormone: a potent, endogenous regulator of lws1 and lws2 expression. Results: Our studies indicate that additional transcriptional differences beyond opsin expression exist between LWS1 and LWS2 cones. Bulk RNA-Seq results showed 95 transcripts enriched in LWS1 cones and 186 transcripts enriched in LWS2 cones (FC > 2, FDR < 0.05). In situ hybridization results also reveal underlying heterogeneity within the lws1- and lws2-expressing populations. This heterogeneity is evident in cones of mature zebrafish, and further heterogeneity is revealed in transcriptional responses to TH treatments. Discussion: We found some evidence of coordinate regulation of lws opsins and other genes by exogenous TH in LWS1 vs. LWS2 cones, as well as evidence of gene regulation not mediated by TH. The transcriptional differences between LWS1 and LWS2 cones are likely controlled by multiple signals, including TH.

Ultra-rare complement factor 8 coding variants in families with age-related macular degeneration.

Genome-wide association studies have uncovered 52 independent common and rare variants across 34 genetic loci, which influence susceptibility to age related macular degeneration (AMD). Of the 5 AMD-associated complement genes, complement factor H (CFH) and CFI exhibit a significant rare variant burden implicating a major contribution of the complement pathway to disease pathology. However, the efforts for developing AMD therapy have been challenging as of yet. Here, we report the identification of ultra-rare variants in complement factors 8A and 8B, two components of the terminal complement membrane attack complex (MAC), by whole exome sequencing of a cohort of AMD families. The identified C8 variants impact local interactions among proteins of C8 triplex in vitro, indicating their effect on MAC stability. Our results suggest that MAC, and not the early steps of the complement pathway, might be a more effective target for designing treatments for AMD.

Development of multilineage adult hematopoiesis in the zebrafish with a runx1 truncation mutation.

Runx1 is required for the emergence of hematopoietic stem cells (HSCs) from hemogenic endothelium during embryogenesis. However, its role in the generation and maintenance of HSCs during adult hematopoiesis remains uncertain. Here, we present analysis of a zebrafish mutant line carrying a truncation mutation, W84X, in runx1. The runx1(W84X/W84X) embryos showed blockage in the initiation of definitive hematopoiesis, but some embryos were able to recover from a larval "bloodless" phase and develop to fertile adults with multilineage hematopoiesis. Using cd41-green fluorescent protein transgenic zebrafish and lineage tracing, we demonstrated that the runx1(W84X/W84X) embryos developed cd41(+) HSCs in the aorta-gonad-mesonephros region, which later migrated to the kidney, the site of adult hematopoiesis. Overall, our data suggest that in zebrafish adult HSCs can be formed without an intact runx1.

In vitro modeling and rescue of ciliopathy associated with IQCB1/NPHP5 mutations using patient-derived cells.

Mutations in the IQ calmodulin-binding motif containing B1 (IQCB1)/NPHP5 gene encoding the ciliary protein nephrocystin 5 cause early-onset blinding disease Leber congenital amaurosis (LCA), together with kidney dysfunction in Senior-Løken syndrome. For in vitro disease modeling, we obtained dermal fibroblasts from patients with NPHP5-LCA that were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into retinal pigment epithelium (RPE) and retinal organoids. Patient fibroblasts and RPE demonstrated aberrantly elongated ciliary axonemes. Organoids revealed impaired development of outer segment structures, which are modified primary cilia, and mislocalization of visual pigments to photoreceptor cell soma. All patient-derived cells showed reduced levels of CEP290 protein, a critical cilia transition zone component interacting with NPHP5, providing a plausible mechanism for aberrant ciliary gating and cargo transport. Disease phenotype in NPHP5-LCA retinal organoids could be rescued by adeno-associated virus (AAV)-mediated IQCB1/NPHP5 gene augmentation therapy. Our studies thus establish a human disease model and a path for treatment of NPHP5-LCA.

Differential requirement for Gata1 DNA binding and transactivation between primitive and definitive stages of hematopoiesis in zebrafish.

The transcription factor Gata1 is required for the development of erythrocytes and megakaryocytes. Previous studies with a complementation rescue approach showed that the zinc finger domains are required for both primitive and definitive hematopoiesis. Here we report a novel zebrafish gata1 mutant with an N-ethyl-N-nitrosourea-induced point mutation in the C-finger (gata1(T301K)). The Gata1 protein with this mutation bound to its DNA target sequence with reduced affinity and transactivated inefficiently in a reporter assay. gata1(T301K/T301K) fish had a decreased number of erythrocytes during primitive hematopoiesis but normal adult hematopoiesis. We crossed the gata1(T301K/T301K) fish with those carrying the R339X mutation, also known as vlad tepes (vlt), which abolishes DNA binding and transactivation activities. As we reported previously, gata1(vlt/vlt) embryos were "bloodless" and died approximately 11 to 15 days after fertilization. Interestingly, the gata1(T301K/vlt) fish had nearly a complete block of primitive hematopoiesis, but they resumed hematopoiesis between 7 and 14 days after fertilization and grew to phenotypically normal fish with normal adult hematopoiesis. Our findings suggest that the impact of Gata1 on hematopoiesis correlates with its DNA-binding ability and that primitive hematopoiesis is more sensitive to reduction in Gata1 function than definitive hematopoiesis.

A Novel ARL3 Gene Mutation Associated With Autosomal Dominant Retinal Degeneration.

Despite major progress in the discovery of causative genes, many individuals and families with inherited retinal degenerations (IRDs) remain without a molecular diagnosis. We applied whole exome sequencing to identify the genetic cause in a family with an autosomal dominant IRD. Eye examinations were performed and affected patients were studied with electroretinography and kinetic and chromatic static perimetry. Sequence variants were analyzed in genes (n = 271) associated with IRDs listed on the RetNet database. We applied a stepwise filtering process involving the allele frequency in the control population, in silico prediction tools for pathogenicity, and evolutionary conservation to prioritize the potential causal variant(s). Sanger sequencing and segregation analysis were performed on the proband and other family members. The IRD in this family is expressed as a widespread progressive retinal degeneration with maculopathy. A novel heterozygous variant (c.200A > T) was identified in the ARL3 gene, leading to the substitution of aspartic acid to valine at position 67. The Asp67 residue is evolutionary conserved, and the change p.Asp67Val is predicted to be pathogenic. This variant was segregated in affected members of the family and was absent from an unaffected individual. Two previous reports of a de novo missense mutation in the ARL3 gene, each describing a family with two affected generations, are the only examples to date of autosomal dominant IRD associated with this photoreceptor gene. Our results, identifying a novel pathogenic variant in ARL3 in a four-generation family with a dominant IRD, augment the evidence that the ARL3 gene is another cause of non-syndromic retinal degeneration.

WT1 induces apoptosis through transcriptional regulation of the proapoptotic Bcl-2 family member Bak.

Wilms' tumor or nephroblastoma is believed to arise from embryonic nephrogenic rests of multipotent cells that fail to terminally differentiate into epithelium and continue to proliferate. The WT1 tumor suppressor gene, a transcription factor controlling the mesenchymal-epithelial transition in renal development, is mutated in 10% to 15% of Wilms' tumors. This potentially explains the disordered differentiation and proliferation program of a subset of Wilms' tumors. To elucidate the role of mutations of WT1 in the etiology of Wilms' tumor, we used an inducible cellular system for expressing wild-type and tumor-derived missense mutant WT1 proteins. Expression of wild-type WT1, but not mutant proteins, blocked cellular proliferation and DNA synthesis and rapidly induced apoptosis. We showed that wild-type WT1 induced transcription of one of the seven studied proapoptotic genes, Bak. Furthermore, WT1 protein bound to specific DNA-binding sites located in the Bak promoter and Bak was critical to WT1-mediated apoptosis, as overexpression of VDAC2, a specific Bak inhibitor, attenuated WT1-mediated cell death. These data support the hypothesis that Wilms' tumors arise, in part, because WT1 mutant proteins fail to promote programmed cell death during kidney development.

In Vitro Modeling Using Ciliopathy-Patient-Derived Cells Reveals Distinct Cilia Dysfunctions Caused by CEP290 Mutations.

Mutations in CEP290, a transition zone protein in primary cilia, cause diverse ciliopathies, including Leber congenital amaurosis (LCA) and Joubert-syndrome and related disorders (JSRD). We examined cilia biogenesis and function in cells derived from CEP290-LCA and CEP290-JSRD patients. CEP290 protein was reduced in LCA fibroblasts with no detectable impact on cilia; however, optic cups derived from induced pluripotent stem cells (iPSCs) of CEP290-LCA patients displayed less developed photoreceptor cilia. Lack of CEP290 in JSRD fibroblasts resulted in abnormal cilia and decreased ciliogenesis. We observed selectively reduced localization of ADCY3 and ARL13B. Notably, Hedgehog signaling was augmented in CEP290-JSRD because of enhanced ciliary transport of Smoothened and GPR161. These results demonstrate a direct correlation between the extent of ciliogenesis defects in fibroblasts and photoreceptors with phenotypic severity in JSRD and LCA, respectively, and strengthen the role of CEP290 as a selective ciliary gatekeeper for transport of signaling molecules in and out of the cilium.

Despite WT1 binding sites in the promoter region of human and mouse nucleoporin glycoprotein 210, WT1 does not influence expression of GP210.

BACKGROUND: Glycoprotein 210 (GP210) is a transmembrane component of the nuclear pore complex of metazoans, with a short carboxyterminus protruding towards the cytoplasm. Its function is unknown, but it is considered to be a major structural component of metazoan nuclear pores. Yet, our previous findings showed pronounced differences in expression levels in embryonic mouse tissues and cell lines. In order to identify factors regulating GP210, the genomic organization of human GP210 was analyzed in silico. RESULTS: The human gene was mapped to chromosome 3 and consists of 40 exons spread over 102 kb. The deduced 1887 amino acid showed a high degree of alignment homology to previously reported orthologues. Experimentally we defined two transcription initiation sites, 18 and 29 bp upstream of the ATG start codon. The promoter region is characterized by a CpG island and several consensus binding motifs for gene regulatory transcription factors, including clustered sites associated with Sp1 and the Wilms' tumor suppressor gene zinc finger protein (WT1). In addition, distal to the translation start we found a (GT)n repetitive sequence, an element known for its ability to bind WT1. Homologies for these motifs could be identified in the corresponding mouse genomic region. However, experimental tetracycline dependent induction of WT1 in SAOS osteosarcoma cells did not influence GP210 transcription. CONCLUSION: Although mouse GP210 was identified as an early response gene during induced metanephric kidney development, and WT1 binding sites were identified in the promoter region of the human GP210 gene, experimental modulation of WT1 expression did not influence expression of GP210. Therefore, WT1 is probably not regulating GP210 expression. Instead, we suggest that the identified Sp binding sites are involved.

Knockdown of Bardet-Biedl syndrome gene BBS9/PTHB1 leads to cilia defects.

Bardet-Biedl Syndrome (BBS, MIM#209900) is a genetically heterogeneous disorder with pleiotropic phenotypes that include retinopathy, mental retardation, obesity and renal abnormalities. Of the 15 genes identified so far, seven encode core proteins that form a stable complex called BBSome, which is implicated in trafficking of proteins to cilia. Though BBS9 (also known as PTHB1) is reportedly a component of BBSome, its direct function has not yet been elucidated. Using zebrafish as a model, we show that knockdown of bbs9 with specific antisense morpholinos leads to developmental abnormalities in retina and brain including hydrocephaly that are consistent with the core phenotypes observed in syndromic ciliopathies. Knockdown of bbs9 also causes reduced number and length of cilia in Kupffer's vesicle. We also demonstrate that an orthologous human BBS9 mRNA, but not one carrying a missense mutation identified in BBS patients, can rescue the bbs9 morphant phenotype. Consistent with these findings, knockdown of Bbs9 in mouse IMCD3 cells results in the absence of cilia. Our studies suggest a key conserved role of BBS9 in biogenesis and/or function of cilia in zebrafish and mammals.

Wnt signaling gradients establish planar cell polarity by inducing Vangl2 phosphorylation through Ror2.

It is fundamentally important that signaling gradients provide positional information to govern morphogenesis of multicellular organisms. Morphogen gradients can generate different cell types in specific spatial order at distinct threshold concentrations. However, it is largely unknown whether and how signaling gradients also control cell polarities by acting as global cues. Here, we show that Wnt signaling gradient provides directional information to a field of cells. Vangl2, a core component in planar cell polarity, forms Wnt-induced receptor complex with Ror2 to sense Wnt dosages. Wnts dose-dependently induce Vangl2 phosphorylation of serine/threonine residues and Vangl2 activities depend on its levels of phosphorylation. In the limb bud, Wnt5a signaling gradient controls limb elongation by establishing PCP in chondrocytes along the proximal-distal axis through regulating Vangl2 phosphorylation. Our studies have provided new insight to Robinow syndrome, Brachydactyly Type B1, and spinal bifida which are caused by mutations in human ROR2, WNT5A, or VANGL.

Definitive hematopoietic stem/progenitor cells manifest distinct differentiation output in the zebrafish VDA and PBI.

One unique feature of vertebrate definitive hematopoiesis is the ontogenic switching of hematopoietic stem cells from one anatomical compartment or niche to another. In mice, hematopoietic stem cells are believed to originate in the aorta-gonad-mesonephros (AGM), subsequently migrate to the fetal liver (FL) and finally colonize the bone marrow (BM). Yet, the differentiation potential of hematopoietic stem cells within early niches such as the AGM and FL remains incompletely defined. Here, we present in vivo analysis to delineate the differentiation potential of definitive hematopoietic stem/progenitor cells (HSPCs) in the zebrafish AGM and FL analogies, namely the ventral wall of dorsal aorta (VDA) and the posterior blood island (PBI), respectively. Cell fate mapping and analysis of zebrafish runx1(w84x) and vlad tepes (vlt(m651)) mutants revealed that HSPCs in the PBI gave rise to both erythroid and myeloid lineages. However, we surprisingly found that HSPCs in the VDA were not quiescent but were uniquely adapted to generate myeloid but not erythroid lineage cells. We further showed that such distinct differentiation output of HSPCs was, at least in part, ascribed to the different micro-environments present in these two niches. Our results highlight the importance of niche in shaping the differentiation output of developing HSPCs.

Methods for reverse genetic screening in zebrafish by resequencing and TILLING.

Animal models provide an in vivo system to study gene function by transgenic and knockout approaches. Targeted knockout approaches have been very successful in mice, but are currently not feasible in zebrafish due to the inability to grow embryonic stem cells. As an alternative, a reverse genetic approach that utilizes screening by resequencing and/or TILLING (Targeting Induced Local Lesions INGenomes) of mutagenized genomes has recently gained popularity in the zebrafish field. Spermatogonia of healthy males are mutagenized using ENU (N-ethyl-N-nitrosourea) and F1 progeny is collected by breeding treated males with healthy wild type females. Sperm and DNA banks are generated from F1 males. DNA is screened for ENU-induced mutations by sequencing or TILLING. These mutations can then be studied by in vitro fertilization (IVF) from the cryopreserved sperm of the corresponding F1 male followed by breeding to homozygosity. A high-throughput method of screening for rare heterozygotes and efficient recovery of mutant lines are important in identification of a large number of mutations using this approach. This article provides optimized protocols for resequencing and TILLING based on our experiences. We performed a pilot screen on 1235 F1 males by resequencing 54 exons from 17 genes and analyzed the sequencing data using multiple programs to maximize the mutation detection with minimal false positive detection. As an alternative to sequencing, we developed the protocols for TILLING by capillary electrophoresis using an ABI Genetic analyzer 3100 platform followed by fragment analysis using GeneScan and Genotyper softwares. PCR products generated by fluorescently labeled universal primers and tailed exon-specific primers were pooled 4-fold prior to heteroduplex formation. Overall, our pilot screen shows that a combination of TILLING and sequencing is optimal for achieving cost-effective, high-throughput screening of a large number of samples. Amplicons with fewer common SNPs are ideal for TILLING whereas amplicons with multiple SNPs and in/del polymorphisms are best suited for sequencing followed by analysis with SNPdetector.

The receptor tyrosine kinase regulator Sprouty1 is a target of the tumor suppressor WT1 and important for kidney development.

WT1 encodes a transcription factor involved in kidney development and tumorigenesis. Using representational difference analysis, we identified a new set of WT1 targets, including a homologue of the Drosophila receptor tyrosine kinase regulator, sprouty. Sprouty1 was up-regulated in cell lines expressing wild-type but not mutant WT1. WT1 bound to the endogenous sprouty1 promoter in vivo and directly regulated sprouty1 through an early growth response gene-1 binding site. Expression of Sprouty1 and WT1 overlapped in the developing metanephric mesenchyme, and Sprouty1, like WT1, plays a key role in the early steps of glomerulus formation. Disruption of Sprouty1 expression in embryonic kidney explants by antisense oligonucleotides reduced condensation of the metanephric mesenchyme, leading to a decreased number of glomeruli. In addition, sprouty1 was expressed in the ureteric tree and antisense-treated ureteric trees had cystic lumens. Therefore, sprouty1 represents a physiologically relevant target gene of WT1 during kidney development.